A new 85 kDa, breast tumour associated antigen — A potential diagnostic and prognostic agent

In an attempt to develop measures for early diagnosis and prognosis of the disease and to explore association of murine mammary tumour virus (MuMTV) or related virus in breast cancer, we purified a breast tumour associated antigen (BTAA) from the breast tumour tissues of untreated female cancer patients. The BTAA purified by DEAE discontinuous column chromatography followed by SE-HPLC was an 85 kDa glycoprotein. A high level of circulating antibodies against this antigen was observed, using ELISA, in all the untreated female breast cancer patients. The BTAA was not immunologically related to MuMTV antigens but strongly resembled an 83 kDa glycoprotein tumour associated antigen, purified from MuMTV induced mouse mammary tumour. In patients after surgical removal of the breast tumour, the circulating antibodies to the BTAA decreased gradually, but reappeared in the patients with secondary metastasis. In healthy age matched women or in female patients with carcinoma of tissues other than breast, no significant titre of the BTAA antibodies was observed.     

Humoral antibodies to the antigens related to the structural protein antigens of the mammary cancer virus (MTV) in breast cancer patients and in healthy donors

Indirect radioimmunoassay and immunoperoxidase studies provided further evidence of human serum reactivity with murine mammary tumor virus (MMTV) structural proteins. Examination of over 400 human sera from breast cancer (BC) patients and controls has shown that the incidence of antibodies which react with MMTV structural proteins was significantly higher in BC patients (50%) than in patients with carcinomas of other organs (3%) or normal women (3%). However, the percentage of subjects immune to MMTV was higher in pregnant women (10%) as compared with normal subjects. All the women with BC of the first clinical stage had antibodies to MMTV. The percentage of immune donors was lower among patients with BC of the later stages (IIa, 90%, IIb, 76%, III, 27%, IV, 0%). Thus, a good agreement has been demonstrated between the first stages of BC and expression of antibodies to MMTV.     

Antibodies to MMTV-related antigen in breast cancer patients of different ages

Summary The proportion of women with breast cancer in whom circulating antibodies to mouse mammary tumor virus (MMTV)-related antigen were detected by indirect immunofluorescence was found to increase with advancing age of patients. The incidence of patients with breast cancer in whom these antibodies were not detected increased up to age 40; in older patients it was essentially independent of age. This observation suggests that the higher incidence of breast cancer among older women in the general population correlates with a subset of patients in whom circulating antibodies to MMTV can be demonstrated.     

High risk human papillomavirus and Epstein Barr virus in human breast milk

ABSTRACT: BACKGROUND: Multiple viruses, including human immunodeficiency virus, Epstein Barr virus (EBV) and mouse mammary tumour virus have been identified in human milk. High risk human papillomavirus (HPV) sequences have been identified in breast cancer. The aim of this study is to determine if viral sequences are present in human milk from normal lactating women. FINDINGS: Standard (liquid) and in situ polymerase chain reaction (PCR) techniques were used to identify HPV and EBV in human milk samples from normal lactating Australian women who had no history of breast cancer.High risk human papillomavirus was identified in milk samples of 6 of 40 (15%) from normal lactating women - sequencing on four samples showed three were HPV 16 and one was HPV 18. Epstein Barr virus was identified in fourteen samples (33%). CONCLUSION: The presence of high risk HPV and EBV in human milk suggests the possibility of milk transmission of these viruses. However, given the rarity of viral associated malignancies in young people, it is possible but unlikely, that such transmission is associated with breast or other cancers.     

清洁级实验动物四种病毒抗体玻片酶免疫检测试剂盒的研制

本文报道对清洁级实验动物应排除的四种病毒（淋巴细胞脉络丛脑膜炎病毒、小鼠脱脚病病毒、鼠肝炎病毒和仙台病毒）抗体玻片酶免疫（ＥＩＡ）检测试剂盒的研制。四种病毒感染的细胞和对照细胞经冷丙酮固定于载玻片上制成特异性抗原和对照抗原,此四种病毒的抗血清各１０份和ＳＰＦ小鼠血清２０份分别与四种病毒的特异性抗原和对照抗原进行ＥＩＡ交叉试验,结果显示,抗原只与其相应抗血清发生特异性显色反应,与非特异性小鼠血清和ＳＰＦ小鼠血清不显色。与ＨＩ或ＥＬＩＳＡ方法比较,通过对１１２份普通小鼠血清进行测试,结果表明,ＥＩＡ对仙台病毒抗体的检出率（１９．６％）显著高于（＜０．００５）ＨＩ（６．３％）,对小鼠脱脚病病毒抗体的检出率（２３．３％）与ＨＩ（２１．４％）无显著性差异（Ｐ＞０．０５）。ＥＩＡ对淋巴细胞脉络丛脑膜炎病毒和鼠肝炎病毒抗体的检出率分别为１．８％和７１．２％,ＥＬＩＳＡ对两种病毒抗体的检出率分别为１．８％和６７．６％,两种方法对两种病毒抗体的检出率无显著性差异（Ｐ＞０．０５）。重复性试验表明两批四种病毒抗体试剂盒对１０８份小鼠血清两次测定的符合率为９６～１００％。四种病毒的ＥＩＡ抗原在－１８℃保存１２个月或在２－８℃保存３     

An Antigen in Human Breast Cancer Sera related to the Murine Mammary Tumour Virus

THE possibility of an oncogenic virus in human breast cancer has been increased by recent findings. Virus-like particles resembling the mammary tumour virus (MTV) were found in electron micrographs of human breast cancer tissue¹ and particles physically and morphologically similar to MTV particles have been seen in human milk². These particles were found more frequently in the milk of American women with a history of breast cancer in their immediate families and in the milk of Parsi women in Bombay than in the milk of nonselected American women³. Parsi women are three times more likely to have breast cancer than other women in Bombay³. The detection of RNA-dependent DNA-polymer-ase activity in such particles isolated from human milk emphasized the possibility that these particles represent an oncogenic RNA virus⁴. In addition to the physical and morphological resemblance of human milk particles and MTV an immunological relationship between these two kinds of particles seems probable; sera from breast cancer patients neutralize the biological activity of MTV whereas normal human sera did not do so⁵. We report data supporting the hypothesis of an immunological cross relationship between MTV and human breast cancer.     

Lymphocyte transformation in vitro to RII mouse milk antigen among woman with breast disease.

The cell-mediated immune responses of 110 women with benign or malignant breast disease were tested in in vitro lymphocyte transformation assay with an antigen preparation made from RIII mouse milk containing mammary tumor virus. About 50% of patients responded positively to the milk preparation. In contrast, 25% of normal women or women with other gynecological malignancies responded positively to the antigen (P = 0.015). The data demonstrate a similar response pattern among women with malignant or benign breast disease. In addition, a subpopulation of normal women with positive response to this antigen is clearly defined.     

Calorie consumption level influences development of C3H/Ou breast adenocarcinoma with indifference to calorie source

To analyze simultaneously the influence attributable to calorie consumption level and percentage of dietary fat on the spontaneous development of mammary adenocarcinoma, virgin female C3H/Ou mice were separated into five dietary groups. Four groups of mice were fed purified diets either ad libitum (16-18 kcal/mouse/day) or restricted 40% in calorie consumption (10-11 kcal/mouse/day), and diets contained either 4.5%, 7.5%, 67%, or 68% calories from fat. Mice that consumed isocaloric diets developed breast malignancy at a comparable pace. Consuming a diet in which fats were present only at levels sufficient to satisfy the threshold requirement of essential fatty acids, 4.5-7.5% of the total calories, or alternatively where dietary fat represented greater than 67% of the total calories consumed, did not significantly alter the tendency for breast tumor development. The pace and frequency with which tumors occurred reflected the host's level of calorie consumption. Mice consuming a high caloric diet, low or high in fat, tended to have a shortened latency to breast tumor formation, an increased incidence of breast tumors, elevated serum prolactin levels, elevated levels of antibodies to mouse mammary tumor virus, and elevated circulating immune complex levels.     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.     

Seromonitoring of laboratory mouse and rat colonies for common murine pathogens (author's transl)]

During a period from 1973 to 1978, 392 and 225 lots including 12,232 mouse and 8,044 rat individual sera, respectively, were examined for antibodies to murine hepatitis virus, Sendai virus, Bordetella bronchiseptica, Mycoplasma pulmonis, Tyzzer agents, Salmonella typhimurium and Corynebacterium kutscheri. Of mouse lots 94.5% and 39.3% from breeder and user colonies, respectively, were negative for all antibodies examined as well as 31.6% and 17.2% of rat breeder and user colonies, respectively. Among positive lots from mouse users, high positivity rates were seen with Senai virus (47.6%), M. pulmonis (19.0%), and murine hepatitis virus (JHM : 18.2%, MHV : 31.0%), while the rates were high in rat user lots with Sendai virus (24.4%), B. bronchiseptica (39.3%) M. pulmonis (12.5%), murine coronaviruses (JHM : 19.0%, MHV-2 : 28.0%) and tyzzer agents (MSK : 19.6%, RT : 17.9%). These pathogenes with high positivities should be monitored indispensably as a quality control of laboratory mice and rats.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

High-mannose glycans are elevated during breast cancer progression

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.     

Mouse immunoglobulin antibodies require complement for neutralization of mouse retroviruses.

The addition of guinea pig complement was found to enhance the neutralizing capacity of mouse antibodies directed against the endogenous ecotropic murine leukemia viruses. The same immune sera, when tested without complement, had low or negligible neutralizing capacities, regardless of whether freshly harvested, unfrozen virus was used to preserve virus infectivity. Antibodies in high titers were found in sera from NFS congenic mice carrying the mouse leukemia virus inducing locus Akv-2. These mouse antibodies were type specific and failed to neutralize either Friend or Moloney leukemia virus. The mouse serum immunoglobulin fraction containing the complement-dependent antibodies was tentatively identified as immunoglobulin M.     

Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gp160 humoral immune response in HIV type 1-infected chimpanzees

The fine specificities of antibodies produced against human immunodeficiency virus type 1 (HIV-1) gp160 were examined in sera from 23 HIV-1-infected chimpanzees. These animals had been infected with one of six isolates of HIV-1. Sera were screened by enzyme-linked immunosorbent assay for reactivity against seven synthetic peptides corresponding to regions of gp160. Chimpanzees appear to remain healthy after infection with HIV-1, suggesting that these animals may prevent extensive spread of the virus in vivo through immunologic mechanisms. Antibody specificity to gp160 epitopes may play a key role in the defense against HIV-1-related disease. Approximately one-half of all chimpanzee sera contained antibodies reactive with peptide 846-860, which corresponds to the carboxyl terminus of gp41. Less than 10% of sera from HIV-1-infected humans that were examined contained antibodies reactive with peptide 846-860, suggesting that this region is not highly immunogenic in humans. Of the human sera containing antibodies reactive with this peptide, all were from individuals classified as Walter Reed stages 1 to 3. No sera from humans with advanced stages of the disease contained antibodies reactive with peptide 846-860. Peptide 600-611, which reportedly reacts with nearly all sera from HIV-infected humans, was reactive with less than one-half of sera from HIV-1-infected chimpanzees. The observed differences in antibody reactivity to gp160 peptides in sera from HIV-1-infected chimpanzees and humans suggest that each may generate antibodies against differing sets of HIV-1 epitopes. These differences may contribute to the lack of disease progression in chimpanzees after infection with HIV-1.     

Autoimmune thyroid diseases in women with breast cancer and colorectal cancer

The aim of the study was to compare the prevalence of autoimmune thyroid diseases in three groups of women (66 with breast cancer (CaB), 68 with colorectal cancer (CaC) and 49 without oncological diseases as a control group). Serum levels of thyroid-stimulating hormone (TSH), free thyroxin (fT4), antibodies to thyroglobulin (TGB-ab) and thyroperoxidase (TPO-ab) and tumor markers CEA, CA 15-3 and CA 19-9 were investigated in all subjects by using the chemiluminiscence method. In contrast to Graves' disease (no observed case), autoimmune thyroiditis was diagnosed in 24.2 % women with CaB (4.5 % euthyroid and 19.7 % with subclinical or overt hypothyroidism), compared to 16.7 % in women with CaC (2.0 % euthyroid and 14.7 % with subclinical or overt hypothyroidism) and 16.2 % controls (4.0 % euthyroid and 12.2 % with subclinical or overt hypothyroidism). Serum levels of TGB-ab were higher in the group with breast cancer as compared to those with colorectal cancer and the control group (medians: 35.80 vs. 31.75 vs. 27.70, p<0.001). Similarly, the percentage of positive TGB-ab and TPO-ab serum levels was higher in women with breast cancer as compared to those with colorectal cancer and the control group. The results of the study support the controversial theory that there is an increased prevalence of autoimmune thyroiditis in women with breast cancer.     

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally syngeneic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 -chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV⁺ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargetin T cells with bispecific antibodies against syngeneic breast cancer.